Medical composition containing photocrosslinkable chitosan derivative

ABSTRACT

A medical composition is provided which is advantageous as a sealant for wound openings as well as have a function promoting healing of intractable wound or incision wound, and which accelerate tissue regeneration but does not cause any side effect such as canceration. The present invention relates to a medical composition comprising a photo-crosslinkable chitosan derivative and a wound healing promoter. The photo-crosslinkable chitosan derivative is preferably a polymer obtainable by incorporating a carbohydrate chain containing a reducing terminal to at least one portion of amino groups of chitosan back bone and incorporating a photoreactive group to at least another part of the amino groups. The wound healing promoter is preferably a growth factor.

TECHNICAL FIELD

The present invention relates to a medical composition comprising aphoto-crosslinkable chitosan derivative and a wound healing promoter,more particularly to a composition combining adhesive activity and woundhealing promoting activity, capable of adhering to an intractable skindisease such as a diabetic skin ulcer or a pressure sore, or a wound,and healing it by appropriately sustained-releasing the wound healingpromoter; and a method of manufacturing said composition having saidphoto-crosslinkable chitosan derivative as the base agent; andapplications of said composition.

BACKGROUND ART

Wounds in skin and the like are mainly categorized into open wounds inwhich normal skin and mucosa tissues are separated and opened, bumscaused by heat or radiation, ulcers (circumscribed tissue defect),complexed wounds thereof, and the like. In particular, for a severewound having a wide area and a deep depth, treatment that preventsexogenous inflammation due to infections and promotes the derivation ofgranulation tissues is essential.

For the treatment of such wounds, wound dressings such as gauze havelong been used. However, since wound dressings themselves have nohealing effects, innovations have been made to improve healing effectsby concurrently using drugs such as antibiotics. Recently, due toadvances in materials chemistry, transparent adhesive films andgelatinous hydrogels that prevent water intrusion while having gaspermeability, and further, artificial skin-like dressings utilizingbiological membranes from animals or the like have come to be used.However, nothing has yet been provided that contains both protectionfunctions such as wound protection and defense against infection, andhealing functions that promote healing at the same time.

In particular, in intractable wounds such as pressure sores or skinulcers derived from diabetes, since blood circulation in defectivetissues is inhibited, regeneration of the defective tissues issignificantly delayed. In the present state of affairs, curative drugs,dressings and the like that are effective for the promotion of healinghave not been provided, so there is a risk that severe exogenousinfections may develop concomitantly during the long period required forhealing, and in cases where infections are concomitantly developed, inorder to treat them, there is no choice but to conduct surgicalprocedures for which there is a large burden on the patient, such as theroot-and-branch excision of necrotic tissues.

For the treatment of such intractable wounds, the use of regeneratedtissues obtained by culturing tissue cells in vitro has been considered,but problems such as convenience, cost, time, safety and the like arestill left. Recently, research on treatment methods using genes thatpromote vascularization in order to induce tissue regeneration has beenadvanced, but many problems have yet to be resolved, such as themethodology for gene transfer and whether or not proteins produced bytransgenes have clinical effects.

Further, other than the abovementioned kinds of wounds, there aresurgical excision wounds from the treatment of organs such as thestomach or the lungs, and for such wounds, dressings having a highadhesiveness are used as sealants for preventing the leakage of air orbody fluids in the anastomotic parts of tissues. For example,biologically-derived protein and cyanoacrylate-based syntheticadhesives, and further, human-derived plasma formulations such as bloodcoagulation factors are used. However, in the present state of affairs,their adhesiveness, safety, biodegradability and the like are notsufficient. In particular, it is important to induce tissue regenerationin incisions and having proactive healing promoting effects, in order tocontribute largely to the prognosis of patients, but a practical sealanthaving such functions has yet to be provided.

Meanwhile, it is known that growth factors (GF) control the propagationand differentiation of cells, and contribute to tissue regeneration. Inparticular, research on the fibroblast growth factors FGF-1 and FGF-2has been advancing, and it has become apparent that these control thepropagation, migration, differentiation, life span and the like ofcells, and contribute to fetal development, vascularization, boneformation, nerve formation, and wound repair. However, it is difficultto maintain and store the activity of GF, and generally, GF interactswith heparin molecules in the proteoglycan constituting theextracellular matrix, thereby protection against deteriorization fromoxidation and function adjustment is done. Further, when concentrated GFacts at one time, there is a risk that cancer-like elements might beelicited, so it must be used carefully. That is, though it has beenexpected that the tissue regeneration function of GF could be utilizedfor wound treatment, its actual clinical application has been hinderedby the problems of GF itself, such as high diffusivity and rapidfunctional deterioration.

The inventors of the present invention have been developing atherapeutic agent for wounds utilizing a polysaccharide, chitosan (referto WO00/27889). Chitosan had been known as a material combining bothwound healing effects and antibacterial activity, but since the controlof water solubility and gelation was difficult, it was difficult to useconventionally as a multipurpose and functional adhesive or dressing. Wehave improved by chemical modification the physical characteristics ofchitosan, which solubilized only in acid regions, so that it is readilysoluble in neutral and alkaline regions, and at the same time,introduced a photoreactive group that can functionally makesintermolecular crosslinking. The photo-crosslinkable chitosan derivativethereby obtained forms a viscous aqueous solution in physiological pH,and said aqueous solution becomes a contact type insoluble gel underirradiation by light. Therefore, it has become evident that an aqueoussolution of the photo-crosslinkable chitosan derivative which wasdeveloped, can be applied to any affected area, and can immediatelyproduce an adhesive gel by photoreaction at said area, and therefore maybe used effectively as an adhesive for wounded areas.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a medicalcomposition which promotes tissue regeneration and at the same time isfree from the risk of causing side effects such as canceration, throughadding the function of proactively promoting the healing of intractablewounds and surgical incision wounds to the composition containing thephoto-crosslinkable chitosan derivative we developed that has theadvantageous functions as a sealant for wounds such as (1) strong andquick adhesiveness, (2) healing characteristics, (3) safety, and (4)appropriate biodegradability.

Therefore, the present invention provides a medical compositioncharacterized in that it contains the photo-crosslinkable chitosanderivative and a wound healing promoter.

In addition to possessing advantageous characteristics such as theforegoing (1) through (4) as a sealant (medical adhesive) for woundregions, this composition is prepared in physiological pH, so that woundhealing promoters having physiological activity, such as GF, can befavorably incorporated without being denatured. The obtained compositioncan be applied to any wound region, and becomes an occlusive andinsoluble gel matrix by merely irradiating said region with light. Thecrosslinked gel matrix obtained from said photo-crosslinkable chitosanderivative not only rigidly holds wound treatment promoters such asgrowth factors, but also sustained-releases the held GF and the like atan appropriate rate. Due to this appropriate sustained-releasecharacteristic, vascularization induction effects can be realized over along period without canceration of tissues by high doses of growthfactor. As a result, healing of intractable wounds and incision woundscan be promoted.

Further, the present invention also provides a composition concurrentlyholding glycosaminoglycans such as heparin, which are important forfunctional control of GF. Differently from the conventional basicchitosan, in which controlling the formation of a polyion complex withacidic heparins is difficult, the photo-crosslinkable chitosanderivative used in the present invention can easily hold heparins, andcan improve GF function at the affected area by interaction withheparin.

Therefore, the present invention also provides a crosslinked chitosanmatrix manufactured by irradiating a composition containing thephoto-crosslinkable chitosan derivative and the wound healing promoter,and containing glycosaminoglycans if desired. This matrix can be used asa drug releasing body for releasing wound healing promoters such asgrowth factors and heparins held therein at an appropriate rate. Inparticular, the matrix holding the growth factor can be also used as acell culture medium for tissue regeneration, due to the activity of thereleased growth factor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing a light absorption spectrum for aqueoussolutions of lactose introduced chitosan derivative (CH-LA), visiblelight reactive compound (FPP) and visible light crosslinkable typechitosan derivative (VL-RC).

FIG. 2 is a graph showing in vitro release of low-molecular dyes (trypanblue and toluidine blue) from the crosslinked chitosan matrix of thepresent invention to PBS.

FIG. 3 is a graph showing in vitro release of polymer substances(polysaccharides and proteins) from the crosslinked chitosan matrix ofthe present invention to PBS.

FIG. 4 is a graph showing in vitro release of polymer substances (FGF-2)from the crosslinked chitosan matrix of the present invention to PBS.

FIG. 5 is a graph showing the relationship between the molecular densityof crosslinked chitosan matrix (chitosan derivative concentration) andretention ratio of substances held therein.

FIG. 6 is a graph showing the relationship between the growth of HUVECon the crosslinked chitosan matrix of the present invention and releaseof FGF-2 from the matrix.

FIG. 7 is a graph showing in vivo release of low-molecular dye (trypanblue) from the crosslinked chitosan matrix of the present invention.

FIG. 8 is a graph showing angiogenesis effect by sustained release ofgrowth factor (FGF) from the crosslinked chitosan matrix of the presentinvention. This demonstrates the relationship between the held FGFconcentration and the amount of hemoglobin. ◯ indicates FGF-carryingchitosan matrix, and ● indicates matrix further containing heparin.

FIG. 9 is a graph showing angiogenesis effect by sustained release ofgrowth factor (FGF) from the crosslinked chitosan matrix of the presentinvention. This demonstrates the time-dependent change of the amount ofhemoglobin. ◯ indicates FGF-carrying chitosan matrix, ● indicates matrixfurther containing heparin, ♦ indicates a comparative example carryingonly heparin, and Δ indicates aqueous solution of UV-RC supplementedwith FGF.

FIG. 10 is a photograph showing healing effect of the crosslinkedchitosan matrix of the present invention in a wound model formed indb/db mouse. (A) indicates untreated control, (B) indicates a woundcoated with crosslinked chitosan matrix without FGF, and (C) indicates awound coated with crosslinked chitosan matrix of the present inventionfurther containing FGF.

FIG. 11 is a graph showing healing effect of the crosslinked chitosanmatrix of the present invention in a wound model formed in db/db mouse.(A) indicates untreated control, (B) indicates a wound coated withcrosslinked chitosan matrix without FGF, and (C) indicates a woundcoated with crosslinked chitosan matrix of the present invention furthercontaining FGF.

FIG. 12 is a photograph showing healing effect of the crosslinkedchitosan matrix of the present invention in a wound model formed in db/+mouse. (A) indicates untreated control, (B) indicates a wound coatedwith crosslinked chitosan matrix without FGF, and (C) indicates a woundcoated with crosslinked chitosan matrix of the present invention furthercontaining FGF.

FIG. 13 is a graph showing healing effect of the crosslinked chitosanmatrix of the present invention in a wound model formed in db/+ mouse.(A) indicates untreated control, (B) indicates a wound coated withcrosslinked chitosan matrix without FGF, and (C) indicates a woundcoated with crosslinked chitosan matrix of the present invention furthercontaining FGF.

FIG. 14 is a photograph showing time-dependent change of wound area in awound model formed in db/db mouse. (A) indicates untreated control, (B)indicates a wound coated with crosslinked chitosan matrix without FGF,and (C) indicates a wound coated with crosslinked chitosan matrix of thepresent invention further containing FGF.

FIG. 15 is a microscopic photograph of a sample obtained by stainingwith HE the cross-section of the wound at fourth day in a wound modelformed in db/db mouse. (A) indicates untreated control, (B) indicates awound coated with crosslinked chitosan matrix without FGF, and (C)indicates a wound coated with crosslinked chitosan matrix of the presentinvention further containing FGF.

FIG. 15 is a microscopic photograph of a sample obtained by stainingwith CD34 the cross-section of the wound at fourth day in a wound modelformed in db/db mouse. (A) indicates untreated control, (B) indicates awound coated with crosslinked chitosan matrix of the present inventionfurther containing FGF.

BEST MODE FOR CARRYUNG OUT THE INVENTION

The photo-crosslinkable chitosan derivative usable in the medicalcomposition of the present invention preferably has a structure in whicha photo-crosslinkable group and a carbohydrate chain are introduced intoa polymer back bone, which is generally called as chitin/chitosan. Inparticular, those formed by incorporating a carbohydrate having reducingterminals and a photo-reactive functional group to at least a portion ofthe 2-position amino groups in the glucosamin units constituting an atleast partially deacetylated chitin/chitosan are preferable.

Normally, chitin/chitosans are deacetylated acid-soluble fractionsobtained by alkali processing chitin (poly-N-acetylglucosamins)originated from crab shells, and generally have the constituent unitsexpressed by the following formulas (1) and (2).

Among chitin/chitosans, some persons call those having a low degree ofdeacetylation (normally less than 40%) as “chitins” and those having ahigh degree of deacetylation (normally 40% or more) as “chitosans”, buthenceforth in the present specification, all chitin/chitosans which areat least partially deacetylated shall be referred to collectively as“chitosans”. Additionally, in the present invention, chitosans are notlimited to those of natural origin, and may be chemically modifiedcarbohydrate chains having similar structures synthesized chemically orby genetic engineering.

Here, “degree of deacetylation” refers to the proportion of acetylaminogroups in the 2-position of the carbohydrate units constituting thechitosan (or poly-N-acetylglucosamin), which have been converted to freeamino groups by deacetylation. In the present specification, the degreeof deacetylation is measured by means of the “colloidal titrationmethod” described in “Health Foods Standard and Criterion (No. 4)”,Japan Health Food and Nutrition Food Association (1996), p. 55.

The chitosan derivative of the present invention has been functionalizedby further chemically modifying the chitosan, and the chitosan used asthe raw material should preferably have a degree of deacetylation of atleast 40%, preferably 60-100%, more preferably 65-95%. A chitosan havinga 100% degree of acetylation consists entirely of the constituent unitsof the above-given formula (1), and does not include the constituentunits of formula (2).

Additionally, there are no particular restrictions on the molecularweight of the chitosan, and this can be changed of a wide rangedepending on the projected use of the chitosan derivative, but ingeneral, the number-average molecular weight should be in the range of5,000-2,000,000, preferably 10,000-1,800,000, more preferably40,000-1,500,000.

The chitosan derivatives suitable for the present invention are thoseformed by incorporating a carbohydrate having reducing terminals to atleast a portion of the 2-position amino groups in the glucosamin units(1) constituting the above-described chitosan and a photo-reactivefunctional group to at least another portion of the 2-position aminogroups. Details of such chitosan derivatives are described inWO00/27889.

The carbohydrates having reducing terminals to be incorporated to thechitosan derivatives include aldoses and ketoses, among which thosehaving 20 or less constituent carbohydrate units, especially those with1-7 units are preferably used. Specific examples include pentaoses andhexaoses such as glucose, fructose, galactose, fucose, mannose,arabinose, xylose, erythrose, hepturose and hexylose, aminocarbohydrates such as glucosamin, N-acetylglucosamin and galacsamin;carbohydrate derivatives such as uronic acids and deoxysaccharides; di-and trisaccharides such as maltose, isomaltose, lactose, melibiose andmaltotriose composed of carbohydrate chains combining theabove-mentioned monosaccharides; and the various oligosaccharides, amongwhich the neutral disaccharides such as maltose, lactose and melibioseare preferable.

While it is also possible to derive chitosans from organic compoundssuch as polyethers and polyhydric alcohols instead of theabove-mentioned carbohydrates, it is preferable to use naturalcarbohydrate chains in consideration of biocompatibility.

The incorporation of the above-mentioned carbohydrates in the 2-positionamino group of the glucosamin units of the chitosan of the above-givenformula (1) can itself be performed using known methods. For example,methods of carboxylating the reducing terminal of a carbohydrate, thenbinding to the 2-position amino group by an amide bond (see, forexample, Japanese Patent Application, First Publication No. H10-120705),or of aldehydating or carbonylating the reducing terminal of acarbohydrate, then binding to the 2-position amino group of a glucosaminunit by a reduction alkylation method by means of a Schiff base (see,for example, “Applications of Chitins and Chitosans”, edited byChitin/Chitosan Workshop, pp. 53-56, Feb. 20, 1990, published by GihodoShuppan K K).

The carbohydrate incorporated in the chitosan in the present inventionis not limited to only one type, and it is possible to use a combinationof 2 or more.

Specific examples of a carbohydrate side chain constituting the chitosanderivative of the present invention include the following, but there isno restriction to these.

-   (i) Carbohydrate derived from lactose:-   (ii) Carbohydrate derived from maltose:-   (iii) Carbohydrate derived from melibiose:-   (iv) Carbohydrate derived from cellobiose:-   (v) Carbohydrate derived from laminalibiose:-   (vi) Carbohydrate derived from mannobiose:-   (vii) Carbohydrate derived from N-acetylchitobiose:

Of the carbohydrate side chains given in the above (i)-(vii), those onthe left side represent residual groups incorporated by means ofcondensation between a carboxyl group on the carbohydrate and a2-position amino group on the chitosan, while those on the right siderepresent residual groups bound by a Schiff base.

While the degree of substitution of 2-position amino groups in theglucosamin units of chitosan by carbohydrate side chains can be changeddepending on the physical properties desired in the final chitosanderivative, the degree of substitution should generally be in the rangeof 0.1-80%, preferably 0.5-60%, more preferably 1-40%. Here, the “degreeof substitution” of the carbohydrate side chain is the level to whichthe amino groups in the 2-position of the carbohydrate unitsconstituting the chitosans are substituted by carbohydrate side chains,and denote the proportion of substituted amino groups with respect tothe total number of free amino groups and substituted amino groups atthe 2-position of the carbohydrate units constituting the chitosans. Inthe present specification, the degree of substitution of carbohydrateside chains is measured by the “phenol-sulfuric acid method” wherein thecharacteristic color emission due to a reaction between carbohydratechains and phenol in sulfuric acid is sensed by light absorption at 490nm (see J. E. Hodge, B. T. Hofreiter, “Methods in CarbohydrateChemistry”, ed. by R. L. Whistler, M. L. Wolfrom, vol. 1, p. 388,Academic Press, New York (1962)).

The chitosan derivative of the present invention has a self-crosslinkingproperty by photo-irradiation due to incorporating photo-reactivefunctional groups in the 2-position amino groups in the glucosamin unitsof the above-given formula (1) constituting the chitosan.

The photo-reactive functional groups used for chemical modification ofthe chitosans according to the present invention are groups which reactwith each other and/or amino groups or hydroxyl groups present in thechitosan upon irradiation by ultraviolet light including thenear-ultraviolet region of 200-380 nm to form crosslinking bondsincluding, for example, those derivable from cyclic unsaturatedcompounds such as benzophenones, cinnamic acids, azides, diolefins andbis-anthracene, especially preferable being those having carbonylazidegroups, sulfonylazide groups and aromatic azide groups.

The photo-reactive group may be a substitutional group which reacts byirradiation of visible light of about 400 to 500 nm. Suchvisible-light-reactive groups include, for example, formyl styryl grouprepresented by the following formula and described in Journal of PolymerScience: Polymer Chemistry Edition, Vol. 20, 1419-1432 (1982).

(In this formula, Ar denotes a heterocyclic ring such as pyridin,alkylpyridinium salt, quinolin, or alkylquinolinium salt.)

The incorporation of photo-reactive functional groups to the aminogroups at the 2-position in the glucosamin units of the chitosans canitself be performed by known methods, for example, by a method ofbinding an azide compound having a carboxyl group to the 2-positionamino group in the presence of a condensing agent (see Japanese PatentApplication, First Publication No. H10-120705); or a method of reactingthe azide compound with the 2-position amino group by means of an acidchloride group, an aldehyde group, an N-hydroxysuccinic acid imide estergroup or an epoxy group (see “Applications of Chitins and Chitosans”,edited by Chitin/Chitosan Workshop, pp. 53-5645-65, Feb. 20, 1990,published by Gihodo Shuppan K K). In azide group crosslinking reactions,it has been conventionally held to be effective to use polyfunctionalcompounds such as bis-azides or above (see Japanese Patent Application,First Publication No. H9-103481), this is not necessary in the presentinvention, so that a chitosan derivative having adequateself-crosslinking ability can be obtained by incorporation of monoazidecompounds.

Specific examples of a photo-reactive group forming the chitosanderivative of the present invention include, for example, thoseexpressed by the following formulas (A) through (E). The group offormula (A) is derived from p-azidobenzoic acid, the group of formula(B) is derived from p-azidobenzaldehyde, the group of formula (C) isderived from p-benzoylbenzoic acid, the group of formula (D) is derivedfrom cinnamic acid, and the group of formula (E) is derived from1-methyl-4-[2-formylphenyl]ethenyl]pyridinium.

While the degree of substitution of these photo-reactive functionalgroups can be changed according to the degree of gelification(insolubility) due to the crosslinking reaction desired in the finalchitosan derivative, but it is preferable for the degree of substitutionof the photo-reactive functional groups to be within the range of0.1-80%, preferably 0.5-50%, more preferably 1-30%. Here, the “degree ofsubstitution” of the photo-reactive functional groups is the degree ofsubstitution of the 2-position amino groups of the carbohydrate unitsforming the chitosans with photo-reactive functional groups, and is theproportion of substituted amino groups with respect to the total numberof free amino groups and substituted amino groups at the 2-position ofthe carbohydrate units forming the chitosans. In the presentspecification, the degree of substitution of photo-reactive functionalgroups such as azide groups can be determined based on calibrationcurves obtained from characteristic absorption at 270 nm for4-azidobenzoic acid.

The degree of substitution of the total of carbohydrate side chains andphoto-reactive functional groups in the chitosan derivatives of thepresent invention is not particularly restricted, and may vary over aconsiderable range, but is usually in the range of 0.2-80%, preferably1.5-65%, more preferably 3-50%.

Additionally, according to the present invention, a hydrogel withconsiderably improved water retention ability can be obtained byincorporating an amphipathic group to at least a portion of the 3- or6-position hydroxyl groups in the carbohydrate units of formulas (1) and(2), and the amino groups in the 2-position of the carbohydrate units offormula (1) constituting the chitosan. These amphipathic groups aregroups having a hydrophobic block comprising a hydrophobic group and ahydrophilic block comprising a hydrophilic group, and often have asurfactant function. Among these those in which the molecular weightratio between the hydrophobic blocks (X) and the hydrophilic blocks (Y)is X:Y=1:5 to 5:1 are preferably used, and non-ionic groups withoutdissociated ionic groups are more preferably used. In particular, thosecomposed of a hydrophobic alkyl block and a hydrophilic polyoxyalkyleneblock and with a molecular weight of at least 90 are preferable, apolyoxyalkylene alkyl ether of 500-10,000 being more preferable. While apolyether not having a hydrophobic block may be used, a polyoxyalkylenealkyl ether is preferable for having both a hydrophobic block and ahydrophilic block in consideration of the improvement to the waterretaining ability.

The incorporation of these amphipathic groups to the chitosan can beperformed, for example, by a method of incorporating a compound havinggroups capable of reacting with amino groups to form covalent bonds,such as aldehyde groups or epoxy groups to a terminal portion of eitherthe hydrophilic block or hydrophobic block of the amphipathic group,then reacting with the 2-position amino group of the glucosamin of thechitosan, a method of inducing a reaction between a polyoxyalkylenealkyl ether derivative having a carboxyl group with the chitosan in thepresence of a condensing agent, or a method of inducing a reactionbetween a polyoxyalkylene alkyl ether derivative having an acid chloridegroup with a hydroxyl group or amino group in the chitosan.

For example, when incorporating a polyoxyalkylene alkyl ether group withan epoxy group on its terminal into an amino group in the chitosan, theamphipathic group is expressed by the following formula (a), and whenincorporating a polyoxyalkylene alkyl ether group with an aldehyde groupon its terminal into an amino group of the chitosan, the amphipathicgroup is expressed by the following formula (b). Additionally, whenbinding a polyoxyalkylene alkyl ether group with an acid chloride groupon its terminal to the 3- or 6-position hydroxyl group of the chitosan,the amphipathic groups are expressed by the following formula (c). Inthe below formulas (a)-(c), n and m are repeating units numbering 1 ormore.

The degree of incorporation of amphipathic groups in the chitosanderivatives of the present invention is not particularly restricted, butshould be within the range normally of 5-70%, preferably 15-55% based onthe change in weight of the chitosan derivative after incorporation.

As described in detail above, in the photo-crosslinkable chitosanderivative used for the medical composition of the present invention,carbohydrates having a reducing terminus and a photoreactive group maybe introduced into the chitosan backbone structure, and amphipathicgroups can be introduced therein as desired. By introducing thecarbohydrate, the chitosan derivative becomes well soluble in neutralregions, can be made into a solution by a physiological buffer or aculture media, and can be mixed without losing the activity of drugs,such as proteins, that may get denatured by acid or alkali. Further, byintroducing the photoreactive group, an insoluble gel body may be formedimmediately by light irradiation after application to an appropriateregion, which adheres to tissues, and a wound healing promoter may beenclosed therein and sustained-released later.

As the polymer constituting the backbone structure of thephoto-crosslinkable chitosan derivative of the present invention,instead of chitosan, polysaccharides such as hyaluronic acid, proteinssuch as collagen, and other synthetic polymers and the like can be used,but carbohydrates capable of sealing wounds and tissues, having drugholding characteristics and appropriate biodegradability, and having anability to conduct controlled release of a drug at a speed which is nottoo fast are most suitable. Among these, chitosan, which itself haswound healing characteristics and antibiotic action, or carbohydratessuch as hyaluronic acid are preferable, and chitosan is more preferablein view of the supply of raw materials and cost.

Further, it is possible to introduce a group having chemicallycrosslinkable characteristics instead of the photoreactive group. Theintroduced group is preferably capable of rapid intramolecularcrosslinking and easy switching thereof. Since the photoreactive grouphas the characteristics of easy switching, high reactivity, and fewunreacted active sites remain, the photoreactive group can be suitablyused. Further, chitosan, having an amino group at the second position,is also advantageous for the introduction reaction of the photoreactivegroup, and therefore, they are suitably used.

The medical composition of the present invention contains at least onewound healing promoter in addition to the foregoing photo-crosslinkablechitosan derivative. The wound healing promoter in the present inventioncan be a substance capable of promoting healing effects in the woundregion to which said composition is applied in every sense. Examplesthereof include drugs having wound healing effects described in “DrugDirectory Fifth Edition” (Pharmacists Association of Osaka PrefecturalHospital, Yakugyojiho Co., 1992) and its addenda edition (1992). Forexample, antibacterial agents and antibiotics for promoting healing bypreventing infections and inflammation at wound regions, or acesodynesand anesthetic agents to alleviate pains caused by wounds are alsoincluded. In particular, when applied to an intractable skin disorder inwhich blood circulation is inhibited such as a pressure sore or adiabetic skin ulcer, substances inducing vascularization such as growthfactors and angiogenesis factors are preferable. The growth factor usedhere is not particularly limited as long as the growth factor inducesvascularization and has a function to promote granulation. For example,FGF-1, FGF-2, HB-EGF, HGF, VEGF₁₆₅, and HGF can be cited. Further, sincethe sustained-drug-releasing body of the present invention has extremelyhigh tissue adhering characteristics, it is possible to conductvoluntary supply of the growth factor at the wound region byincorporating plasmids containing genes expressing the abovementionedgrowth factors.

Further, the medical composition of the present invention may furthercontain glycosaminoglycans such as heparin and heparan sulfate inaddition to the foregoing photo-crosslinkable chitosan derivative andthe wound healing promoter. For example, the growth factor is consideredto adjust functions by interacting with heparin molecules inproteoglycan constituting the extracellular matrix existing in thevicinity of the wound tissues. Therefore, by letting the composition ofthe present invention contain glycosaminoglycans such as heparin, it ispossible to give the composition of the present invention a function asa source of the glycosaminoglycans. These glycosaminoglycans can besimply mixed in the composition, or can be covalent-bound with thechitosan backbone structure as described in, for example, WO00/27889.

Further, the composition of the present invention may contain othermedically acceptable additives. As examples, interleukin, leukemiainhibitor factor, interferon, TGF-β, erythropoietin, trombopoietin andthe like are included. Further, other drugs known to induce apoptosis inmammals can be used. Examples of such drugs include TNF-α, TNF-β, CD30ligand, and 4-1BB ligand. Further, chemotherapy drugs useful fortreating cancers can be used. Examples of such chemotherapy drugsinclude an alkylating agent, folic acid antagonist, metabolic productsof nucleic acids, antibiotics, pyrimidine analogs, 5-fluorouracil,cisplatin, purine nucleoside, amines, amino acid, triazole nucleoside,corticosteroids, and hormone drugs functioning to adjust or inhibithormone action to tumors such as tamoxifen and onapristone.

The medical composition of the present invention can be prepared bydissolving the photo-crosslinkable chitosan derivative, the woundhealing promoter, and other desired components into a solvent,preferably an aqueous medium in preferably neutral pH.

The composition prepared as above preferably has appropriate viscosityconsidering that such composition is applied to wound regions. Forexample, according to a commercially available rotary viscometer (forexample, B type viscometer, manufactured by TOKIMEC Inc. (Tokyo,Japan)), when viscosity is set to about 100 to 10,000 cps (centipoise(mPa·s)), preferably about from 150 to 8,000, and more preferably fromabout 200 to 6,500 cps, application to a vertical wound or a tissue facebecomes easy. Further, when viscosity is set to about from 250 to 5,000cps, and preferably about from 300 to 2,000 cps, it becomes easy forapplication and free from runoff at any wound or tissue face, andholding time becomes an appropriate length, and enough time is allowedfor subsequent procedures such as light irradiation. However, inapplication to a horizontal wound face, filling into a concave portionand the like, a composition having low viscosity of under about 300 cps,further under about 200 cps, even further under about 100 cps can beused. Further, for example, a composition having viscosity as low as,for example, pure water can be applied to regions other than ahorizontal wound face by using a spray device and the like. In operativeendoscopy through catheters or the like, appropriate viscosity can beadopted by considering fluidity in the tube, holding characteristics atthe incised wound due to endoscopic operation, and the like.

That is, content of the photo-crosslinkable chitosan derivative in themedical composition of the present invention is set to at least 3 mg/ml,preferably at least 5 mg/ml, more preferably at least 7.5 mg/ml, muchmore preferably at least 10 mg/ml, and even much more preferably atleast 15 mg/ml in order to secure holding characteristics at appliedregions and securely hold the drugs contained in the matrix aftercrosslinking.

Content of the wound healing promoter is determined as appropriate inorder to sustained-release the quantity required in the applied region.For example, content of the growth factor in the case of application toa skin wound region is set to about 1 to 1,000 μg/ml, preferably about 5to 500 μg/ml, and more preferably about 10 to 200 μg/ml.

Although depending on the amount of the growth factor concurrentlycontained, content of glycosaminoglycan such as heparin is set topreferably about 10 to 5,000 μg/ml, more preferably about 50 to 1,000μg/ml, and much more preferably about 100 to 500 μg/ml. Contents ofother possible components are set to the degree generally used in themedical field, and their most suitable contents can be easily determinedby those skilled in the art.

The medical composition of the present invention contains aphoto-crosslinkable chitosan derivative. Therefore, by irradiating themedical composition of the present invention with light having a givenstrength (ultraviolet light, visible light and the like) for apredetermined time, crosslinking occurs in a short time to form aninsoluble matrix. The formed crosslinked chitosan matrix holds additivessuch as wound healing promoter therein, and can sustained-release theseadditives at an appropriate rate.

Conditions for crosslinking by light vary according to the types anddegree of substitution of the photoreactive groups introduced into thephoto-crosslinkable chitosan derivative to be used, amounts of thechitosan derivative contained in the composition and amounts of the drugto be held, amounts of the composition to be used and desirablesustained-release rates and the like. In general, when about 100 μl of acomposition containing at least about 7.5 mg/ml of thephoto-crosslinkable chitosan derivative is used, light irradiation froma light source provided at about 2 cm from the composition is conductedfor about 0.01 to 300 sec, preferably about 0.05 to 120 sec, and morepreferably about 0.1 to 60 sec, and thereby about 100% crosslinkingdegree of the photoreactive group can be obtained.

By light irradiation, the photo-crosslinkable chitosan derivativecrosslinks and an insoluble matrix is formed. The crosslinked chitosanmatrix formed contains inside wound healing promoters such as growthfactors and other additives such as heparin. These drugs may beinitially released to some extent due to diffusion characteristics ofthe drug itself. However, most of the drug is rigidly held inside thecrosslinked chitosan matrix. After that, when the crosslinked chitosanmatrix is biodegraded, the held drug is sustained-released at anappropriate rate.

The crosslinking reaction degree of the photoreactive group of thechitosan matrix is not particularly limited. In general, it isconsidered that there is a trend that when the crosslinking reactiondegree is high, initial release of the drug is small and suitablefunction as a drug releasing body can be obtained. Therefore, thecrosslinked chitosan matrix of the present invention has a crosslinkingreaction degree of at least 30, preferably from 40 to 100%, morepreferably from 50 to 100%, much more preferably from 60 to 100%, andeven much more preferably from 70 to 100%. Here, “crosslinking reactiondegree (or crosslinking degree)” in the present invention represents theratio of photoreactive groups bound with other groups and the like,among the photoreactive groups existing in the photo-crosslinkablechitosan derivative.

As a more specific usage form, the medical composition of the presentinvention is applied to tissues having, for example, a skin ulcer, andirradiated with light. Since the composition has appropriate viscosity,the composition is held without running off from the damaged region,rapidly becomes an adhesive and insoluble gel body (matrix) due tointermolecular crosslinking by light irradiation, seals the damagedregion and concurrently sustained-releases the contained drug, andaccelerates protection and healing of the wound. Further, in stomachwalls, cancer tissues and the like, by containing the drug to treatthem, treatment effects can be derived as a coating for the stomachulcer and tumor tissues.

Therefore, the crosslinked chitosan matrix formed by irradiating themedical composition of the present invention with light constructs avery superior sustained-drug-releasing body, which is particularlysuitable for healing wounds.

It is astonishing that the basic growth factor (FGF-2) conventionallyconsidered not able to be held within the basic chitosan backbonestructure has been held and sustained-released by the matrix formed fromthe photo-crosslinkable chitosan derivative of the present invention toa degree equal to or more than the acidic growth factor (FGF-1).

Further, when an amphipathic group such as polyoxyalkylene alkylether isfurther introduced into the photocrosslinkable chitosan derivative, thecrosslinked chitosan matrix, after light irradiation, becomes able torapidly absorb lots of moisture close to 100 times its own weight. Inthis case, the crosslinked chitosan matrix can absorb bleeding from thewound region or the surgery region to accelerate hemostasis.

Further, the crosslinked chitosan matrix of the present invention hasthe characteristics that the cell growth factor is held therein, and thecell growth factor is gradually released. Therefore, the crosslinkedchitosan matrix of the present invention can also be used as a cellculture medium material for tissue regeneration. For example, themedical composition of the present invention is applied to the surfaceof the cell culture plate or the like, light irradiation is conducted,and a membranous crosslinked chitosan matrix is formed. When the targetcell culture medium is arranged on the covered part of said cell cultureplate and incubation is conducted, growth of said cells is stimulated.Therefore, in the present invention, the foregoing crosslinked chitosanmatrix, specifically the matrix containing the growth factor can beeffectively used as a cell culture medium material for tissueregeneration.

The composition of the present invention is generally provided as aviscous aqueous solution as described above. However, for example, thecomposition of the present invention can be provided as a solid (forexample powder) obtained by freeze-drying the aqueous solution. Thesolid composition can be promptly used by being dissolved or swollen inan aqueous medium.

EXAMPLES

Detailed descriptions of the present invention will be hereinafter givenby using concrete examples. However, these concrete examples do notlimit the scope of the present invention.

Synthesis Example 1

Synthesis of Photo (Ultraviolet) Crosslinking Chitosan Derivative(UV-RC)

UV-RC wherein an ultraviolet reactive group and a carbohydrate chainwere introduced into a chitosan backbone structure was synthesized inaccordance with the method described in WO00/27889. More specifically,azide (p-azide benzoate) and lactose (lactobionic acid) were introducedby condensation reaction into an amino group of crab-derived chitosanhaving 800 to 1,000 kDa of molecular weight and 80% of deacetylationdegree (available from Yaizu Suisan Industry Co., Ltd.). It wasconfirmed that the resultant was soluble in neutral pH due tointroduction of lactose, and substitution degrees of p-azide benzoateand lactobionic acid were about 2.5% and 2.0% respectively.

Further, when chitosan materials derived from shrimp shell and achitosan material derived from cuttlefish cartilage were used, similarderivatives could be synthesized.

Synthesis Example 2

Synthesis of Photo (Visible Light) Crosslinking Chitosan Derivative(VL-RC)

Methyl-4-[2-(4-formylphenyl)ethenyl]pyridine methosulfonate (FPP)expressed by the following formula was synthesized in accordance withthe method described in Journal of Polymer Science: Polymer ChemistryEdition, Vol. 20, 1419-1432 (1982).

More specifically, under the condition of cooling with ice, a solutionof γ-picolline (3.07 g, 33 mmol) in methanol (8.3 ml) was added todimethyl sulfate (4.16 g, 33 mmol). After the solution was left for 1hour at room temperatures, terephthalic aldehyde (13.4 g, 100 mmol) wasadded to the solution and dissolved therein by heating. Subsequently,piperidine (0.47 ml) was added, and refluxed for 5 hours. A separatedsubstance was removed by heating filtration. A hot filtrate was mixedwith a mixed solvent of ethanol (50 ml) and acetone (16.7 ml), which wasleft overnight at room temperature. A yellow separated substance wasbatched off by filtration, which was washed with ethanol and acetone,and then dried under reduced pressure to obtain FPP. Its yield was 4.81g (46%) and its melting point was from 210 to 213° C.

The derivative wherein lactose was introduced into chitosan as describedin the foregoing Synthesis Example 1 (CH-LA) (1 g) was dissolved indistilled water (100 ml), to which the FPP (368 mg, 1.15 mmol) obtainedby the foregoing was added, and the resultant was stirred for 30minutes. The pH was adjusted to 4.75 by 1N NaOH, and an injectionsolvent (8 ml) of cyano sodium borohydride (112 mg, 1.73 mmol) wasadded. The resultant was stirred for 24 hours at room temperature in thestate of light shielding, which was poured into acetone (360 ml) toobtain a precipitate. The precipitate was sufficiently washed withmethanol and acetone, and dried under reduced pressure to obtain VL-RC.Its yield was 728 mg and its substitution degree was 1.55%.

An aqueous solution containing 0.01 wt % of CH-LA, an aqueous solutioncontaining 0.001 wt % of FPP, and an aqueous solution of 0.01 wt % ofVL-RC obtained in Synthesis Example 2 were prepared. Absorptioncharacteristics of the respective aqueous solutions were measured by afluorescence spectroscopy. A part of these results are shown in FIG. 1by comparison.

As shown in spectrums shown in FIG. 1, regarding VL-RC, the maximumabsorption was shown in the vicinity of from 340 to 350 nm as pure FPP,and it was confirmed that visible light reactive FPP was introduced.Further, when the chitosan material derived from shrimp shell and thechitosan material derived from cuttlefish cartilage were used, thederivatives showing similar absorption in the visible regions could besynthesized as well.

Example 1

2% water solutions of UV-RC and VL-RC obtained in the foregoingSynthesis Examples 1 and 2 were prepared. The respective aqueoussolutions were irradiated with light having various wavelengths by anirradiation device made by Ushio, Inc., and a strength test ofadhesiveness of the crosslinked chitosan matrix was conducted by theprocedure described in Example 4 of WO00/27889.

More specifically, 2 slices of edible ham being 2 mm thick cut in a sizeof 2×3 cm were arranged to obtain a size of 2×6 cm. The foregoingrespective solutions were applied thereto so that an application sizebecame 2×2 cm and 2 mm thick with a central focus on the interfacebetween the two slices of ham.

Immediately after that, the applied solutions were illuminated withlight for 30 sec to bond the two slices of ham. One of the bonded twoslices of ham was fixed to a stand by a clip, an end of the other sliceof ham was progressively weighted, and the weight was measured at whichthe bonded two slices of ham split. Evaluation was made by a weight percross-sectional area of gel (10² kg/m²) when the crosslinking chitosangel bonding the ham was split. The results of the respective derivativeswere relatively shown based on 3×10² kg/m² when UV-RC (crab) was split(Table 1). TABLE 1 Wavelength of irradiation light (nm) Material 245 365400-500 UV-RC (crab) ++ +++ − UV-RC (shrimp) ++ +++ − UV-RC (cuttlefish)+++ +++ − VL-RC (crab) + + +++ VL-RC (shrimp) + + +++ VL-RC(cuttlefish) + ++ +++(−): not insolubilized

Example 2 Holding Characteristics of High Molecular Substance

Holding characteristics of a polymer material (protein) was examined byusing prepared UV-RC derived from crab. In order to change solubilityand holding characteristics of the polymer substance, UV-RC wherein anintroduction ratio of lactose was 0, 2, 5, 10, or 20% was prepared. 1 wt% solution of each UV-RC and 10 wt % bovine serum albumin solution weremixed at a ratio of 4:1 to adjust pH to 6.0 or 7.0. Then, each amount ofan insoluble matter generated in the mixed solution and each viscosityof the mixed solution were compared. The viscosity of the mixed solutionwas estimated from a migration rate of the mixed solution in a glasstube. The results are shown in the following Table 2. TABLE 2 Generationdegree of insoluble matter Viscosity increase (−: not generated) due toadding Lactose introduction ratio Without Albumin albumin of UV-RC (%)pH albumin added (+: increased) 0 6 + + + 7 ++++ +++++ + 2 6 − − + 7++++ +++++ + 5 6 − − − 7 ++ +++ + 10 6 − − − 7 + ++ − 20 6 − − − 7 − ± −

As shown above, when lactose was not introduced, the insoluble matterwas generated in any pH. Meanwhile, by introducing lactose,solubilization under the physiological conditions (pH 6 and/or 7) couldbe achieved. Further, it was found that when the polymer substances suchas protein (albumin) was taken in, the higher the lactose introductionratio was, the harder the insoluble deposit was to generate, that is,the case of the higher lactose introduction ratio is advantageous as aphoto-crosslinking chitosan derivative holding a drug or the like.Further, it was shown that when the lactose introduction ratio was about5% or more, preferably about 10% or more under the physiologicconditions or in the vicinity thereof (pH 6 or 7), lots of polymersubstances such as protein can be suitably held.

Example 3

Drug Release from Crosslinked Chitosan Matrix (1)

An aqueous solution prepared by dissolving 1 mg/ml of trypan blue (modelfor an acid with low molecular weight) or toluidine blue (model for abase with low molecular weight) in a phosphate buffer (PBS) and 2 wt %aqueous solution of UV-RC synthesized from crab-derived chitosan weremixed at a ratio of 1:3. 50 μl of the prepared mixed solution was addedto polyethylene 24-wells tissue culturing multiwell, which wasirradiated with UV to form a crosslinked chitosan matrix layercontaining low molecular weight coloring matter on the bottom face ofthe well. This matrix was washed with PBS, to which 1 ml of PBS wasadded. The resultant was mildly rotated by a rotary shaker at roomtemperature and incubated. PBS was changed every day.

At intervals of given time, PBS was retrieved, and the amount of thecoloring matter eluted into PBS was estimated by absorbance (OD₆₄₀).Time change of residual coloring matter in the crosslinked chitosanmatrix is shown in FIG. 2.

As shown in FIG. 2, the low molecular weight coloring matter held in thecrosslinked chitosan matrix (UV-RC) having a basic amino group wastotally held in a gel over a long period due to ionic interaction in thecase of the acidic trypan blue. However, in the case of the basictoluidine blue, almost all the low molecular weight coloring matter waseluted in PBS within a day.

Example 4

Drug Release from Crosslinked Chitosan Matrix (2)

A test was conducted under the same conditions as in Example 3, exceptthat the low molecular weight coloring matter was changed to thepolysaccharide, heparin, and the proteins, albumin and protamine, andrespective elution characteristics of polymer substances were compared.The amount of eluted heparin was measured by using carbazole assay (Guo,Y., Conrad and H. E., Anal. Biochem., 176, 96-104 (1989), and the amountof protein was measured by using protein assay kits. The results areshown in FIG. 3.

As shown in FIG. 3, heparin and the like, being polymer substances, wereeffectively captured in the crosslinked chitosan matrix, and elutionthereof was controlled over a long period. Heparin, being an acidicpolysaccharide, had the highest holding characteristics; Regarding thebasic protein, about 30% thereof was eluted in a first day, and thenelution was reduced. The neutral protein, albumin, showed behaviorintermediate to both. In any case, significant elution of the heldpolymer substance was not shown on and after the second day. Therefore,it was found that the crosslinked chitosan matrix rigidly holds polymersubstances such as polysaccharides and proteins over a long periodregardless of characteristics such as acidity, neutrality, andalkalinity.

Example 5

Drug Release from Crosslinked Chitosan Matrix (3)

An elution test was conducted as in Examples 3 and 4, except that theheld substance was changed to growth factors, FGF-1, FGF-2, heparinbonding EGF (HB-EGF), and VEGF₁₆₅. Elution amounts of the respectivegrowth factors were estimated by ELISA method (Sigma Aldrich) by usingcommercially available heparinated beads.

In result, elution profiles almost corresponding with those of albuminin Example 4 were observed for all the growth factors. Of the respectivegrowth factors, slight differences were shown in terms of initialelusion on the first day. FGF-1 showed the least elusion amount. Theelusion amount was increased in the order of FGF-2, HB-EGF, and VEGF₁₆₅.However, significant differences among these elution amounts were notshown. In any growth factor, the initial elution amount on the first daywas not beyond 20%. As a representative example, the result in the casethat FGF-2 was held is shown in FIG. 4.

Example 6

Drug Release from Crosslinked Chitosan Matrix (4)

A test similar to that in Example 4 was conducted by using albumin as adrug to be held and changing concentration of UV-RC in the range from0.5 to 1.5 wt %. FIG. 5 shows the result that the albumin amount held inthe crosslinked chitosan matrix after the lapse of the first day isplotted in relation to the UV-RC concentration. As shown in FIG. 5, itis evident that the initial elusion characteristics of the drug,particularly polymer substances, depends on the content of UV-RC, thatis, the molecule density of chitosan.

From the results of Examples 3 to 6, it was demonstrated that polymersubstances such as polysaccharides, protein, and growth factors wereeffectively held in the crosslinked chitosan matrix of the presentinvention. In particular, it was demonstrated that elusion on and afterthe second day was almost totally inhibited, and stable holding wasattained. Further, differently from low molecular substances, these highmolecular substances were well held regardless of moleculecharacteristics such as acidity, neutrality, and basicity. Particularly,it was shown that drugs having lots of acid groups had high holdingstability. Further, in the foregoing proteins and cell growth factors,their elusion inhibitory effects were maintained over 7 days or more.Meanwhile, when PBS was substituted with PBS to whichchitinase/chitosanase mixed enzymes which decompose chitosan hydrogelwas added on the seventh day, elusion of polymer substances wasgradually started. Such sustained-release characteristics due tobiodegradation of the chitosan matrix continued at least 1 week.

Example 7

Culture of HUVEC on Crosslinked Chitosan Matrix

Crosslinked chitosan matrices holding the growth factors (FGF-1, FGF-2,HB-EGF, and VEGF₁₆₅) prepared in Example 4 were formed on a cell culturedish. The matrices were washed with PBS for 1, 3, and 7 days. Afterthat, HUVEC was seeded and cultured for 3 days. After that, growthcharacteristics on the matrices holding the respective growth factorswere evaluated. Further, similar evaluation was made for a culturemedium in which chitinase/chitosanase enzymes were added to a dishcontaining a matrix washed for 7 days. The results are shown in Table 3.The numerical values in the table are relative values where the growthcharacteristic in the case where culture was made on a matrix havingonly UV-RC (no growth factor) was set to 100. TABLE 3 Washing days ofUV-RC (day(s)) 0 1 3 7 7 + Enzyme FGF-1 132 ± 14 126 ± 19 104 ± 18 100 ±19 129 ± 14 FGF-2 173 ± 27 142 ± 11 104 ± 12  97 ± 17 178 ± 27 VEGF₁₆₅159 ± 14 151 ± 17 115 ± 20 107 ± 13 173 ± 21 HB-EGF 109 ± 12 104 ± 14108 ± 17 104 ± 21 112 ± 17

Washing day 0 means that HUVEC was seeded without washing thecrosslinked chitosan matrix. As evidenced by Table 3, in the case ofnon-washing in which there was the initial release of the growth factorfrom the matrix, the highest cell growth characteristics were shown.When release of the growth factor was almost totally inhibited afterwashing one or more days, there were some cases wherein only growthcharacteristics equal to that of the matrix containing no growth factor(numerical value: 100) were seen. However, when the enzymes were added,by re-releasing the respective growth factors associated withdecomposition of the crosslinked chitosan matrix, growth characteristicsequal to of the non-washed matrix was recovered. As a representativeexample, the result of the case wherein FGF-2 was held is shown in FIG.6. The vertical axis of the graph of FIG. 6 represents relative valuesof growth characteristics (growth rate), and correlates with the numberof growth cells. A dotted line in FIG. 6 represents a base line of thenumerical value 100.

Example 8

In Vivo Release of Trypan Blue from Crosslinked Chitosan Matrix

In accordance with Example 3, a crosslinked chitosan matrix holdingtrypan blue was prepared. 100 μl of this crosslinked matrix was buriedin a position being 2 cm apart from a home of a tail on the back of amouse. After the lapse of a given period of time, the matrix was takenout and washed. Chitosan was decomposed by 100 mM of sodium nitrate, andresidual amounts of trypan blue were measured. The result is shown inFIG. 7.

In the result of Example 3, trypan blue was hardly released into PBSfrom the crosslinked chitosan matrix (FIG. 2). Meanwhile, in the case ofin vivo release, as shown in FIG. 7, the chitosan matrix was decomposedby enzymes existing in the body, trypan blue was gradually released overabout two weeks, and the amount of the residual coloring matter in thematrix was decreased.

Example 9

In Vivo Release of Growth Factor from Crosslinked Chitosan Matrix

In accordance with Example 5, a composition containing FGF-1 or FGF-2was prepared, and each 100 Mm thereof was irradiated with UV to obtainthe crosslinked chitosan matrix. Further, the crosslinked chitosanmatrix in which heparin having a final concentration of 20 μg/mlcoexisted in order to stabilize the growth factor was also prepared.These crosslinked chitosan matrices were buried into the back of a mousesimilarly to in Example 8 and left for 4 days. After that, 1×1 cm ofskin around the buried matrix was peeled, skin tissues were finely cut,and red cell hemoglobin in the tissues was extracted by a hemolyticagent (Sigma Aldrich). From the amount of hemoglobin detected by ahemoglobin assay kit (Sigma Aldrich), blood capillary derivationcharacteristics in the region was estimated. The results are shown inFIG. 8. Amounts of the growth factor added to the composition werechanged as 1, 4, and 15 μg/100 μg.

As shown in FIG. 8, it was found that by making 4 μg or more of FGF beheld in the crosslinked chitosan matrix, hemoglobin (Hb) associated withangiogenesis in the vicinity of the region in which the gel was buriedwas significantly increased. Such effects were further intensified byadding heparin which could be expected to provide the effect to maintainthe growth factor by ionic interaction and to improve the function ofFGF.

As a control, burying a chitosan derivative which contains the growthfactor but is not provided with photo-crosslinked was conductedconcurrently. However, in this case, increase of hemoglobin was notfound. It can be considered that the reason thereof was the growthfactor was diffused and released in a short time, and therefore,sustained-release effects could not be obtained. Further, when onlyheparin was held in the crosslinked chitosan matrix, Hb amount was thesame degree as in the case that FGF was not added.

Further, FGF concentration was fixed to 4 μg, and a similar test wascontinued for 15 days. The increase trend of hemoglobin was maintained.As shown in FIG. 9(B), particularly when FGF-2 was used, maintenanceeffects of Hb increase trend was significant. After 15 days, 1 mg ormore level was maintained. Signs of canceration were never observed inthe vicinity of the buried part.

Example 10

Healing Acceleration Effects in Wound Model

(1) Formation of Skin Wound Model in Mouse

In this experiment, C57BL/ksj db/db, which was a female diabetes variantmouse and a normal mouse of its littermate (db/+) (CREA Japan Co.) wereused. All mice were provided with standard experimental diet and water,and used for the experiment when they became over ten weeks old. Beforethe experiment, urine of the mice was checked with respect to glucoseand protein by using a test specimen (Uro-Labstrix: Bayer Medical Co.).In result, it was diagnosed that the db/db mouse had severe diabetes,and db/+ mouse was normal.

Each mouse was given anesthetic by diethyl ether. After back hair wascompletely shaven, a circular wound (about 100 mm²) over a full skinthickness was formed on the upper part of the back of each mouse byusing edged scissors and a surgical knife.

(2) Wound Healing Acceleration Effects

100 μl of a composition in which human recombinant FGF-2 (Pepro Tech ECCo.) was added to 20 mg/ml of UV-RC solution, or a composition in whichFGF-2 was not added was applied to the wound of each mouse. The woundwas irradiated with ultraviolet rays being 2 cm apart from the wound for20 sec to obtain the crosslinked chitosan matrix. A mouse in which asimilar wound (about 100 mm²) was formed to which no treatment wasconducted was used as a control. Change of the wound area of each mousewas measured every two days by using a caliper rule. According to visualobservation, the crosslinked chitosan matrix became a hydrogel hydratedthrough the healing process.

Regarding the db/db mouse, photographs of the wound on the second,fourth, eighth, and 14th days from forming the wound are shown in FIG.10. Further, wound occlusion rates were evaluated as a function betweenratio (%) of non-occlusion area in relation to the area in forming thewound and time. The result is plotted in FIG. 11.

As evidenced by FIGS. 10 and 11, in the control wound (A), only about50% of the wound area was occluded even on the 14th day. It was on andafter 20th day when about 80% of wound occlusion was attained.Meanwhile, the area of the wound covered by the crosslinked chitosanmatrix holding FGF-2 (C) shrunk very rapidly. About on the sixth totenth day, about 80% of the wound was occluded, and on the 14th day, thewound was almost completely healed. Further, the wound covered by onlythe crosslinked chitosan matrix (not containing FGF-2) (B) showed aresult intermediate to both. It was confirmed that the healing effectsby the composition containing the photo-crosslinkable chitosanderivative of the present invention were, in particular, significantlydemonstrated during the initial 2 days, and such effects were continueduntil healing was attained.

Meanwhile, regarding the db/+ mouse, photographs of the wound on thesecond, fourth, and tenth days from forming the wound are shown in FIG.12. The graph of wound healing rates is shown in FIG. 13.

In the case of the normal db/+ mouse, wound healing was faster than inthe db/db mouse having a healing disorder under all conditions. However,when covered with the crosslinked chitosan matrix of the presentinvention, healing was significantly accelerated compared to theuntreated control. More specifically, in the control (A), about 70% ofwound occlusion was shown on the tenth day. Meanwhile, in the case ofcovering with the crosslinked chitosan matrix ((B) and (C)), about 80%of wound occlusion was attained within eight days. Further, healingacceleration effects by adding FGF-2 was significant in the first stageof the healing process (within about six days). After that, nosignificant difference due to the adding of FDF-2 was seen. It can beconsidered that the reason thereof was in the normal db/+ mouse, growthfactors and the like required for healing were provided in vivo as well.That is, the crosslinked chitosan matrix of the present inventioncapable of sustained-releasing a growth factor such as FGF-2appropriately and over a long period of time is particularly suitablefor treatment of intractable wounds such as diabetic skin ulcers.

Healing promotion effects equal to or more than the foregoing could befound when heparin (swine small bowel-derived, Scientific ProteinLaboratories Co.) was further added.

Example 11 Histological Observation of Wound Model in db/db Mouse

Part of skin containing wound tissues was taken on each measurement dayin Example 10. The taken sample was fixed in 10% formaldehyde solution,embedded in paraffin, and sliced in 4 μm thick in a sectionperpendicular to the wound surface (Yamato Kohki Co.). The sliced piecewas put on a glass slide, and stained with hemotoxylin-eosin (HE)reagent.

FIG. 14 shows photographs showing each piece on the second, fourth,eighth, and 16th day. Arrows shown in the figure indicate edges ofepithelialized tissues. In the untreated control (A), epithelializationdid not proceed even on the 16th day. However, in the wound covered withthe chitosan matrix holding FGF-2 (C), epithelialization rapidlyproceeded, and almost complete epithelialization was attained on the16th day In the chitosan matrix containing no FGF-2 (B),epithelialization corresponding to medium between the both was observed.

Photomicrographs of the pieces on the fourth day from forming the woundare shown in FIG. 15. The symbol ▾ affixed in the figure indicates theedge of formed granulation tissues, the white arrow indicates theangiogenesis region, and the black arrow indicates the remainingchitosan matrix. In the wound covered with the chitosan matrix holdingFGF-2 (C), angiogenesis and granulation of tissues proceeded. In thecase of covering with the chitosan matrix containing no FGF-2 (B),slight angiogenesis was also observed. However, in the untreated control(A), any of angiogenesis and granulation could not be observed.

For example, there is a report that when an aqueous solution of FGF-2was added to a wound opening of the db/db mouse once a day, only slighteffects could be shown in terms of acceleration of re-epithelialization(Tsuboi, R. and Rifkin, D. B., J. Exp. Med., 1990: 172, 245-251). Theinventors thereof and the like infer that when the aqueous solution ofFGF-2 was used, a large wound cap was formed at the wound opening, whichadversely affected ambulato of keratinocyte. According to the presentinvention, the wound opening was covered with chitosan matrix(hydrogel). Therefore, it can be considered that a large wound cap wasnot formed, and FGF-2 in the matrix accelerated re-epithelialization.

Further, FIG. 16 shows photomicrographs of the samples wherein the pieceon the fourth day from forming the wound was provided with immunestaining with CD34. It was observed that compared to the untreatedsample (A), in the sample covered with the chitosan matrix holdingFGF-2, vascularization was significantly increased. The results ofaverage numbers of blood vessels existing per one visual field of amicroscope are shown in the following Table 4. TABLE 4 Day Fourth day16th day Control 18.2 33.4 Chitosan matrix 15.1 31.5 FGF-2/chitosanmatrix 37.4 41.6

Example 12

The abdomen of a rat was cut, and an ulcer model was formed on thestomach wall thereof with a laser knife. Healing acceleration effectssimilar to the foregoing was confirmed by pathological tissueobservation. More specifically, the healing rate was improved in orderof the crosslinked chitosan matrix (without GF) and the crosslinkedchitosan matrix+GF. This suggests that even in the nonphysiologicalenvironment by stomach acid (about pH2), chitosan gel protected drugssuch as GF to some extent, and contributed to healing. Ultimately, in aplurality of animal experiments, healing was confirmed in up to about ahalf period compared to the case without covering with the crosslinkedchitosan matrix. However, practically, when used on the stomach wallwhich is under a low pH environment due to stomach acid, it ispreferable that application is made under coexistence with substancesinhibiting action and secretion of stomach acid such as a buffer and H2blocker. However, in digestive organs on and after the intestine, GF andthe like can be well protected and superior wound healing accelerationeffects can be demonstrated without coexistence with such substances.

The foregoing effects were shown not only in using the ultravioletcrosslinking type (UV-RC), but also in using the visible lightcrosslinking type VL-RC similarly

INDUSTRIAL APPLICABILITY

The crosslinking chitosan derivative used in the present invention hasboth a lactose part and a photoreactive group. Therefore, thecrosslinking chitosan derivative is soluble in water in physiological pHin the vicinity of neutrality. By irradiating an aqueous solutionthereof with light (UV or visible light), the aqueous solution becomesan insoluble hydrogel having a strength equal to that of soft rubbergenerally within 10 sec. The formed crosslinked chitosan matrix occludeswounds well, and protects and shrinks the wound in an appropriatehumidified healing environment.

Further, the crosslinked chitosan matrix of the present invention canhold particularly well polymer substances such as polysaccharides,proteins, and growth factors. Along with biodegradation of the matrix invivo, the crosslinked chitosan matrix has an ability tosustained-release the held substance. In particular, when a wound iscovered with the crosslinked chitosan matrix holding a therapeutic agentfor wounds containing a high diffusive growth factor such as FGF, woundhealing is accelerated by appropriately sustained-releasing the growthfactor or the like, and canceration is not induced. Therefore, thecomposition of the present invention is particularly useful forpromoting the healing of intractable wounds in which tissues becomenecrotic such as diabetic skin ulcers.

The crosslinking chitosan derivative of the present invention can beused as a carrier for holding and sustained-releasing a growth factorrelating to wound healing other than FGF such as platelet-derived growthfactor (PDGF), transforming growth factor-β (TGF-β), vascularendothelial cell growth factor (VFGF), and epidermal growth factor(EGF).

Further, the crosslinked chitosan matrix of the present invention can beused as a culture medium material for cell culture or tissueregeneration.

1. Medical composition containing a photo-crosslinkable chitosanderivative which is formed by incorporating to a chitin/chitosancomprising units represented by the following formula (I):

a carbohydrate chain containing a reducing terminal to at least one partof amino groups of the glucosamine units and a photoreactive group to atleast another part of amino groups of the glucosamine units, and a woundhealing promoter.
 2. (canceled)
 3. Composition according to claim 1,characterized in that it contains at least 3 mg/ml of thephoto-crosslinkable chitosan derivative.
 4. Composition according toclaim 1, characterized in that the wound healing promoter is a growthfactor.
 5. Composition according to claim 4, characterized in that thegrowth factor is FGF-1, FGF-2, HB-EGF, VEGF₁₆₅ or HGF.
 6. Compositionaccording to claim 1, characterized in that it further containsglycosaminoglycans.
 7. Composition according to claim 6, characterizedin that the glycosaminogylcans are heparin or heparan sulphate. 8.Crosslinked chitosan derivative matrix obtainable by irradiating lightto the composition according to claim 1 and carrying a wound healingpromoter and optionally glycosaminoglycans.
 9. Crosslinked chitosanderivative matrix according to claim 8, characterized in that itscrosslinking degree by the photoreactive groups is 30 to 100%. 10.Crosslinked chitosan derivative matrix according to claim 8,characterized in that the wound healing promoter is a growth factor. 11.Drug-sustained-release body composed of the crosslinked chitosanderivative matrix according to claim
 8. 12. Base material for tissueregeneration or cell culture comprising the crosslinked chitosanderivative matrix according to claim
 8. 13. Wound dressing for treatmentof an intractable skin disease comprising the composition according toclaim 1.